A method for screening functional anti-Treg antibodies using a Treg-like cell line

Regulatory T cells can suppress activated T cell proliferation by direct cell-contact, although the exact mechanism is poorly understood. Identification of a Treg-specific cell surface molecule that mediates suppression would offer a unique target for cancer immunotherapy to inhibit Treg immunosuppressive function or deplete Tregs in the tumor microenvironment. In this study, we explored a method of whole cell immunization using a Treg-like cell line (MoT cells) to generate and screen monoclonal antibodies that bound cell surface proteins in their native conformations and functionally reversed Treg-mediated suppression. From the 105 hybridomas that bound to the MoT cell surface, a functional screen utilizing conventional Treg suppression assays revealed 32 candidate antibodies that exhibited functional activity (reversed or enhanced suppressive activity). As an example, we characterized one anti-MoT mAb, 12E7, that exhibited strong binding to MoT cells and conventional Treg cell surfaces. This candidate antibody was subsequently found to bind to a potential suppressive target, CD44, and demonstrated the ability to partially reverse MoT and conventional human Treg-mediated suppression.